Formulations for enhanced bioavailability of orally administered polar agents

ABSTRACT

A composition is described having improved oral permeability of polar agents such as neuraminidase inhibitors. The composition includes one or more polar agents and one or more permeability enhancers such that the composition increases the amount of the polar agent capable of being transported across a Caco-2 cell membrane by at least 150% relative to the amount capable of being transported across the Caco-2 Cell membrane in the absence of the permeability enhancer. Oral dosage forms including the composition, and methods of treating or preventing influenza infection are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/001,874 filed Nov. 5, 2007, and 61/030,056 filed Feb. 20, 2008. Thecontents of each of these applications is incorporated by referenceherein, in their entirety and for all purposes.

FIELD OF THE INVENTION

The invention relates generally to enhancing the permeability andbioavailability of active agents. More specifically, the inventionrelates to enhancing the permeability and bioavailability of polaractive agents.

BACKGROUND OF THE INVENTION

Peramivir is a member of a class of antiviral agents that work byinhibiting viral neuraminidase, an enzyme essential for the influenzavirus to replicate and infect its hosts. In addition to influenza A andB, avian influenza virus (H5N1) has been shown to be sensitive toperamivir. More specifically, depending on the influenza strain,peramivir has been determined to be 2 to 10 times more potent as aneuraminidase inhibitor than oseltamivir (Tamiflu®) and zanimivir, basedon experimentally determined concentrations of each required for 50%inhibition of neuraminidase activity in vitro.

It has been reported that studies in rodents and primates haveestablished the safety and efficacy of intramuscular injection ofperamivir in a mouse influenza model. Further intravenous andintramuscular formulations of peramivir are reported to have beenevaluated in pre-clinical animal models with success. However, humanstudies with oral forms of peramivir have demonstrated very poor oralbioavailability of peramivir. In particular it has been reported that ina phase III clinical trial of an oral formulation of peramivir, theantiviral activity against influenza A and B did not reach statisticalsignificance.

Accordingly there is a need for neuraminidase inhibitor compositionswhich exhibit improved bioavailability and efficacy when administeredorally for treatment or prevention of influenza infections.

SUMMARY OF THE INVENTION

The invention features compositions comprising at least one polar agentand at least one permeability enhancer. The compositions can increasethe amount of a polar agent capable of being transported across a cellmembrane such as a Caco-2 cell membrane, and can increase this amount byat least 150% relative to the amount capable of being transported acrossthe cell membrane in the absence of the permeability enhancer. Suitablepermeability enhancers for use in the inventive compositions can befatty acids, fatty acid esters, fatty acid salts, glycerol, glycerolmonocaprylate, surfactants, cyclodextrins, sodium salicylate,ethylenediamine tetraacetic acid, citric acid, chitosan, chitosanderivatives, N-trimethyl chitosan chloride, monocarboxymethyl-chitosan,palmitoyl carnitine chloride, acyl carnitines, ethylene glycoltetraacetic acid, 3-alkylamido-2-alkoxypropyl-phosphocholinederivatives, dimethylpalmityl-ammonio propanesulfonate,alkanoylcholines, N-acetylated amino acids, mucoadhesive polymers,phospholipids, piperine, 1-methylpiperazine, α-amino acids, or mineraloil. Any polar agent that is poorly absorbed by cells can be used in theinventive compositions. In some preferred aspects, the agent is aneuraminidase inhibitor such as oseltamivir, zanamivir, or peramivir.The invention also provides oral dosage forms of the compositions, whichcomprise a therapeutically effective amount of the polar agent and apermeability-enhancing amount of a permeability enhancer. The oraldosage forms can further comprise an enteric- or pH-sensitive coating orlayer surrounding the composition. In the oral dosage forms, thepermeability enhancer can be glycerol, glycerol monocaprylate ordimethylpalmityl-ammonio propanesulfonate. And, the polar agent can be aneuraminidase inhibitor such as oseltamivir, zanamivir, or peramivir.The permeability enhancer can be present in the composition at aconcentration from about 5% to about 95% of the combined weight of thepolar agent and the permeability enhancer.

Also featured are methods for treating or preventing influenzainfection. Generally, the methods comprise administering to a subject inneed thereof a composition comprising at least one neuraminidaseinhibitor and at least one permeability enhancer, including oral dosageforms of such compositions. In the compositions used in the methods, thepermeability enhancer can be glycerol, glycerol monocaprylate ordimethylpalmityl-ammonio propanesulfonate, among others, and theneuraminidase inhibitor can be oseltamivir, zanamivir, or peramivir,among others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of permeability enhancers on peramivir transportacross Caco-2 cell monolayers. Caco-2 cell permeability assays wereconducted according to standard procedures using monolayers having aminimum transepithelial electrical resistance (TEER) of >200 Ωcm2.Except as is indicated each enhancer was present at 1% finalconcentration (w/w) in HBSS. The fold enhancement in transport rate overcontrol (peramivir in HBSS alone) provided by each enhancer is shown.The line indicates a ratio of 1:1 (no enhancement).

FIG. 2 shows the effect of increasing Capmul MCM L8 concentrations onpermeability enhancement of peramivir across Caco-2 cell monolayers.Enhancement saturates at about 1-2% Capmul MCM L8 and declines at higherconcentration. The experiment-to-experiment variation of the maximumfold-enhancement at 1% Capmul MCM L8 shown here and in FIG. 1 isbelieved to be attributed to differences in negative controlpermeability (HBSS alone) between the experiments.

FIG. 3 shows the effect of increasing Gattefosse compositions onpermeability enhancement of peramivir across Caco-2 cell monolayers. Incontrast to results using Capmul MCM L8, enhancement increases arelinear up to at least 2% final concentration, suggesting the maximumpotential enhancement occurs at much higher concentrations.

FIG. 4 shows the effect of increasing PPS concentrations on permeabilityenhancement of peramivir across Caco-2 cell monolayers. A substantiallinear increase in peramivir permeability is observed as the PPSconcentration is increased from 0.001% to 0.01% final concentration.

FIG. 5 shows the effect of increasing glycerol concentrations onpermeability enhancement of peramivir across Caco-2 cell monolayers. Asubstantial increase in peramivir permeability is observed as theglycerol concentration is increased from 2% to 5% final concentration.

DETAILED DESCRIPTION OF THE INVENTION

Peramivir refers to the compound(1S,2S,3S,4R)-3-[(1S)-1-Acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylic acid, and has the chemical structure shown below:

Of particular significance is the presence of three functional groups:an alcohol —OH group, a carboxylic acid group, and a guanidino group.The guanidino group is considered likely to be a major contributor tothe improved activity of peramivir over other agents havingneuraminidase activity. Yet, the poor oral absorption of peramivir andalkyl esters thereof may be due in large part to the highly polar natureof the guanidino group, particularly when in the protonated form such asis found in the zwitterionic form of peramivir. Without intending to belimited to any particular theory or mechanism of action, it is believedthat one or more polar groups on the agent limit permeability of thecompound, and this is particularly problematic where the polar agent isnot or is only weakly transported across the cell membrane by atransport protein. It has now been found that inclusion of one or morepermeability enhancer compounds in formulations with highly polar agentsthat are poorly absorbed, and in particular, neuraminidase inhibitorformulations, can increase the amount of agent that is absorbed bycells, and ultimately increase the bioavailability to the organism. Inparticular, permeability enhancer compound(s) are believed to providepolar agents such as neuraminidase inhibitors with improved oralefficacy with respect to absorption across cellular membranes. Withoutwishing to be bound by any particular theory or mechanism of action, itis believed that a permeability enhancer compound may facilitateincreased absorption of highly polar compounds such as neuraminidaseinhibitors through cellular tight junctions, may act to promoteabsorption through a transcellular pathway, or may act to increasepermeability through other mechanisms. Accordingly, the inventionprovides a composition and method for improving oral bioavailability andactivity of polar compounds such as neuraminidase inhibitors.

“Polar” compounds/agents are those that have at least one group thatconfers a degree of partial or permanent charge on the compound that isgreater than or equal to the charge of a hydroxyl group, more preferablygreater than or equal to the charge of a carboxyl group, more preferablygreater than or equal to the charge of an imidazole group, morepreferably greater than or equal to the charge of an amino group, andmore preferably greater than or equal to the charge of a guanidinogroup, phosphate, or sulfate group.

With respect to a composition aspect of this invention, there isprovided a composition comprising at least one polar agent and apermeability enhancer, wherein the composition provides a Caco-2 polaragent permeability at least 150% of the permeability observed incompositions that do not have a permeability enhancer. As will becomeapparent from the following disclosure and examples, an enhancement inpermeability of 150% is also the equivalent of a 1.5-fold improvement inpolar agent permeability. In preferred aspects, the polar agent is aneuraminidase inhibitor.

Any neuraminidase inhibitor known or discovered in the art can be usedin accordance with the inventive formulations and methods. Non-limitingexamples of such agents includecis-3-[(methylcarbonylamino)methyl]cyclopentanecarboxylic acid;trans-3-amino-c-.sup.4-(methylcarbonylamino)methyl-r-cyclopentanecarboxylicacid;trans-3-{[(amino)(imino)methyl]amino}-c-4-[(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid;4(3-{[(amino)(imino)methyl]amino}-3.alpha.-[(2-hydroxy-1-methylcarbonyl-amino)ethyl)-1-cyclopentanecarboxylicacid; sodium3.beta.-{[amino)(imino)methyl]amino}-4.alpha.-[(2-hydroxy)(1-methylcarbonylamino)ethyl]cyclopentan-r-carboxylate;trans-3-amino-trans-1-hydroxy-cis-4[(hydroxymethyl)(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid;trans-3-{[(amino)(imino)methyl]amino}-trans-1-hydroxy-cis-4-[(2-hydroxymethyl)(1-methylcarbonylamino)ethyl]cyclopentan-r-carboxylicacid;3.beta.-amino-4.alpha.-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclopentancarboxylicacid;3.beta.-{[(amino)(imino)methyl)amino}-4.alpha.-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]-cyclopentancarboxylicacid;cis-3-{[(amino)(imino)methyl]amino)-trans-1-hydroxy-trans-4-[(1-methylcarbonylamino)(2-trifluoromethyl-carbonyloxy)ethyl]cyclopentan-r-carboxylicacid;t-3-amino-c-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl}-t-1-hydroxycyclopentan-r-carboxylicacid;c-3-{[(amino(imino)methyl]amino}-t-1-hydroxy-t-4-{(methylcarbonylamino)([(methyl)-(methoxy)amino]carbonyl}-methyl}cyclopentan-r-carboxylicacid;3.beta.-{[(amino)(imino)methyl]amino}-4.alpha.-{{4-[(methoxy)(methyl)amino]1-(methylcarbonylamino-2-oxo}butyl}cyclopentancarboxylicacid;t-3-{[(amino)(imino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid;t-3-amino-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxy-cyclopentan-r-carboxylicacid;t-3-{[(amino)(imino)methyl]amino}-c-4-[di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-hydroxycyclopentan-r-carboxylicacid;c-3-{[(amino)(imino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid;3.beta.-{[(amino)(imino)methyl]amino}-4.alpha.-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl)cyclopentancarboxylicacid;3.beta.-{[(amino)(imino)methyl]amino}-4.alpha.-[(methylcarbonylamino)(3-pentylaminocarbonyl)methyl]cyclopentancarboxylicacid;3.beta.-{[Amino)(imino)methyl]amino}-4.alpha.-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]cyclopentancarboxylicacid;3.beta.-{1[(Amino)(imino)methyl]amino}-4.alpha.-{[(ethyl)(propyl)aminocarbonyl](methylcarbonylamino)methyl}cyclopentancarboxylicacid;3.beta.-{([(Amino)(imino)methyl]amino}-4.alpha.-{[(ethyl)(propyl)aminocarbonyl](methylcarbonylamino)methyl}cyclopentancarboxylicacid;3.beta.-{[(Amino)(imino)methyl]amino}-4.alpha.-[1-(1-methylcarbonylamino)pent-2-enyl]cyclopentancarboxylicacid;3.beta.-{[(Amino)(imino)methyl]amino}-4.alpha.-[1-(-methylcarbonylamino)pentyl]cyclopentancarboxylicacid; Phenylguanidine; 1-Phenylbiguanide; 4-Acetylaminobenzoic acid;2-Acetylaminobenzenesulfonic acid; 4-Acetylaminophenylphosphoric acid;4-(Trifluoroacetamido)benzoic acid; 4-Thioacetamidobenzoic acid;4-[(Methylsulfonyl)amino]benzoic acid; 3-Guanidinobenzoic acid;3-[Amino(cyanoimino)methyl]aminobenzoic acid; 3-Cyanoaminobenzoic acid;3-(2-Amino-2-imino)ethylbenzoic acid; 4-(Acetamino)phenylacetic acid;4-(Methylaminocarbonyl)benzoic acid;4-Acetylamino-3-hydroxymethylbenzoic acid;beta.-(2-N-Acetylamino-5-carboxyphenyl)ethanol;4-Acetylamino-3-(2′,3′-dihydroxypropyl)benzoic acid;4-Acetylamino-3-aminobenzoic acid;4-Acetylamino-3-[(aminoiminomethyl)amino]benzoic acid;3-[(Aminoiminomethyl)amino]-4-(2-methylpropionylamino)benzoic acid;4-Acetylamino-3-[(hydroxylimino)methyl]benzoic acid;3-[(Aminoiminomethyl)amino]-4-[(methylsulfonyl]amino]benzoic acid;3-[(N-Hydroxyimino)methyl]-4-[(methylsulfonyl)amino]benzoic acid;3-[((Aminoimino)methyl)amino]-4-methoxybenzoic acid;3-[(Aminoiminomethyl)amino]-4-hydroxybenzoic acid;3,5-Bis-[(aminoiminomethyl)amino]benzoic acid;3-Amino-5-{[(aminoimino)methyl]amino}benzoic acid;3-[(Aminoiminomethyl)amino]-5-[(N-hydroxylimino)methyl]benzoic acid; and3-[(Aminoiminomethyl)amino-5-hydroxymethyl)-4-(methylsulfonyl)aminobenzoicacid. Highly preferred neuraminidase inhibitors are(3R,4R,5S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid ethyl ester (oseltamivir),5-acetamido-4-guanidino-6-(1,2,3-trihydroxypropyl)-5,6-dihydro-4H-pyran-2-carboxylicacid (zanamivir), and(1S,2S,3S,4R)-3-[(1S)-1-Acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid (peramivir).

The composition of this invention also contemplates an oral compositioncomprising a therapeutically effective amount of at least one polaragent and a permeability-enhancing amount of a permeability enhancer. Inthis aspect, the enhancing amount of permeability enhancer compound isan amount or concentration which produces a Caco-2 polar agentpermeability at least 150% of (i.e., 1.5-fold over) that provided by thepolar agent in the absence of a permeability enhancer. In some preferredaspects, the polar agent is a neuraminidase inhibitor.

The invention also provides a method for improving the oralbioavailability of polar agents that are not absorbed or only weaklyabsorbed through a cell membrane. Generally, such methods compriseproviding a pharmaceutical formulation comprising a therapeuticallyeffective amount of at least one polar agent and apermeability-enhancing amount of one or more suitable permeabilityenhancer compounds in a pharmaceutical formulation or dosage formthereof which is suitable for oral administration. Examples of suitableforms include, for example, capsules, tablets, caplets, varioussustained or controlled release dosage forms, solutions, suspensions,and the like, each of which may include acceptable pharmaceuticalexcipients which are well known to those skilled in the art and suitablefor formulation of the dosage form in question. In some preferredaspects, the polar agent is a neuraminidase inhibitor. As used herein,the term “permeability enhancer,” “enhancer” and variations thereofrefer to compounds which improve the bioavailability of polar agentswhen incorporated into oral formulations. A neuraminidase inhibitorpermeability enhancer may be defined as a compound capable of increasingthe rate of neuraminidase inhibitor transport across a Caco-2 cellmembrane by 1.5-fold (150%) or more compared to the neuraminidaseinhibitor transport rate in the absence of the enhancer compound. Anymeans known or otherwise available to those of skill in the art can beused to determine the transport rate, including those Caco-2 cellpermeability assays described and exemplified herein.

With respect to the bioavailability of the polar agents, the presence ofa permeability enhancer increases the bioavailability of the agent tothe subject relative to the bioavailability of the agent in the absenceof the permeability enhancer. Thus, in some aspects, the presence of thepermeability enhancer increases bioavailability of the agent about 1.5times the amount of bioavailability of the agent in the absence of thepermeability enhancer, More preferably, the presence of the permeabilityenhancer increases bioavailability of the agent by about 2 times, morepreferably about 2.5 times, more preferably about 3 times, morepreferably about 3.5 times, more preferably about 4 times, morepreferably about 4.5 times, more preferably about 5 times, morepreferably about 6 times, more preferably about 7 times, more preferablyabout 8 times, more preferably about 9 times, more preferably about 10times, more preferably about 12 times, more preferably about 15 times,more preferably about 17 times, more preferably about 20 times, morepreferably about 22 times, more preferably about 25 times, morepreferably about 27 times, more preferably about 30 times or evengreater times the amount of bioavailability of the agent in the absenceof the permeability enhancer.

The invention contemplates that highly polar agents that have lowbioavailability in the absence of a permeability enhancer will haveenhanced bioavailability when combined with a permeability enhancer in aformulation. It is desirable that the bioavailability of the agent beenhanced to at least about 10% in the subject to which the agent isadministered, more preferably at least about 15%, more preferably atleast about 20%, more preferably at least about 25%, more preferably atleast about 30%, more preferably at least about 35%, more preferably atleast about 40%, more preferably at least about 45%, more preferably atleast about 50%, more preferably at least about 55%, more preferably atleast about 60%, more preferably at least about 65%, more preferably atleast about 70%, and more preferably at least about 75% or more in thesubject to which the agent is administered, when formulated with apermeability enhancer.

A variety of classes of compounds may serve as suitable permeabilityenhancers according to the invention. A first category includes fattyacids and salts and esters thereof, including mono-, di-, andtriglycerides. Medium chain length fatty acids, especially C8 and C10acids, and their salts and esters are particularly useful. Suitablespecific examples include sodium caprylate, sodium caprate, CAPMUL®glycerides (available from Abitec of Columbus, Ohio), LABRASOL®glycerides (PEG-8 caprylic/capric glycerides, available from GattefosséSAS of Saint Priest, Cedex, France), GELUCIRE® 44/14 (PEG-32 glyceryllaurate EP, available from Gattefossé), other glycerides & fatty acidesters, CREMOPHOR® (BASF, Ludwigshafen, Germany), D-α-tocopherylpolyethylene glycol 1000 succinate, vegetable oils, polyoxylglycerides,and medium chain mono- and diacylglycerides.

One example of this class, CAPMUL® MCM L8 (glycerol monocaprylate)(available from Abitec of Columbus, Ohio), is composed of mono- anddiglycerides of medium chain fatty acids (mainly caprylic, with somecapric) and 7% maximum free glycerol. It contains at least 44% alphamonoglycerides (as caprylate).

Other examples of this class of enhancers include GATTEFOSSÉcompositions 61A through 61H which are proprietary to Gattefossé SAS,but generally are composed of mixtures containing one or more of mediumchain mono-, di-, or triglycerides, polysorbate derivatives, polyoxylcastor oil derivatives, polyethylene glycol derivatives includingpolyethylene glycol glycerides, polyoxyl ethers, vegetable oils, andsimilar GRAS (generally regarded as safe) lipidic components in varyingamounts. These components are part of individual commercial productssuch as CAPRYOL™ 90, CAPRYOL™ PGMC, LAUROGLYCOL™ 90, GELUCIRE® 44/14,Plurol Oleique CC497, LABRASOL®, LABRAFIL® M1944CS (apricot kernel oilPEG-6 esters), Transcutol HP, Peceol, and Maisine 35-1, all of which areavailable from Gattefossé SAS.

While not falling directly within this class, glycerol itself has beenfound to impart excellent permeability enhancement, particularly forneuraminidase inhibitors. This result was not anticipated as glycerol isnot considered a permeability enhancer.

A second category of enhancers includes surfactants having a steroidalstructure, such as bile acid salts. Examples of suitable compoundsinclude sodium cholate, sodium deoxycholate, glycocholate,glycoursodeoxycholate, taurocholate, taurodeoxycholate, and steroiddetergents/bile salts. Other surfactants may also be suitablepermeability enhancers, including cationic, anionic, and nonionicsurfactants. Examples include polysorbate 80,hexadecyldimethylbenzylammonium chloride, N-hexadecylpyridinium bromide,dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,tetradecyl-β-D-maltoside, octylglucoside, glycyrrhetinic acid,3-(N,N-dimethylpalmitylammonio)propane-sulfonate, and sodium laurylsulfate.

Cyclodextrins may also be used as suitable enhancers. Examples includeβ-cyclodextrin, hydroxypropyl-β-cyclodextrin, γ-cyclodextrin, andhydroxypropyl-γ-cyclodextrin.

A variety of other compounds may also be used as enhancers. Examplesinclude to sodium salicylate, ethylenediamine tetraacetic acid (EDTA),citric acid, chitosan & chitosan derivatives, N-trimethyl chitosanchloride, monocarboxymethyl-chitosan, palmitoyl carnitine chloride, acylcarnitines, ethylene glycol tetraacetic acid (EGTA),3-alkylamido-2-alkoxypropyl-phosphocholine derivatives,alkanoylcholines, N-acetylated amino acids (based on α- and non-α-aminoacids), mucoadhesive polymers, phospholipids, piperine,1-methylpiperazine, α-amino acids, and mineral oil.

Thus a wide variety of enhancer compounds may be selected from the groupconsisting of fatty acids, fatty acid esters, fatty acid salts,glycerol, surfactants, cyclodextrins, sodium salicylate, ethylenediaminetetraacetic acid, citric acid, chitosan, chitosan derivatives,N-trimethyl chitosan chloride, monocarboxymethyl-chitosan, palmitoylcarnitine chloride, acyl carnitines, ethylene glycol tetraacetic acid,3-alkylamido-2-alkoxypropyl-phosphocholine derivatives,alkanoylcholines, N-acetylated amino acids, mucoadhesive polymers,phospholipids, piperine, 1-methylpiperazine, α-amino acids, and mineraloil.

The above examples of permeability enhancers are exemplary only and donot constitute a complete list of potential permeability enhancers. Anycompound capable of increasing the oral absorption of a neuraminidaseinhibitor by at least 50% is considered to be within the scope of thisinvention.

The permeability enhancer and the polar agent may be mixed in anyproportion so long as there is provided a therapeutically effectiveamount of the polar agent and a permeability-enhancing amount of theenhancer compound. Enhancement in bioavailability of orally administeredpolar agents can depend on the nature and concentration of the enhancercompound with which the agent is formulated. It is thus contemplatedthat the required therapeutic amount may be contained in a single dosageform or divided between one or more dosages intended for ingestion atthe same time or in sequence.

The permeability enhancers act relatively independently of theconcentration of polar agent. Differing permeability enhancers can reacheither optimal or maximum enhancement over a wide concentration rangedepending on their particular inherent enhancement potential. Often,enhancers have a non-linear dose response relationship betweenconcentration of enhancer present and amount of increased polar agentabsorption. The amount of enhancer to be utilized in an oral dosage formwith a polar agent is initially based upon the enhancement propertiesobserved in Caco-2 cell assays at varying fixed enhancer concentrations.Based upon those results, an effective in vivo amount of enhancercompound for a human formulation can be estimated, demonstrated andoptimized without undue experimentation using methods well known tothose skilled in the formulation art, to achieve a desiredpharmacokinetic in vivo profile.

In formulating the composition of this invention, it will be apparent tothose skilled in the formulation art that more effective enhancercompounds would require less polar agent than less effectivepermeability enhancers to achieve a target pharmacokinetic profile.Given those considerations and variations, the amount of enhancer may beat least about 0.1 wt % of the combined weight of enhancer and polaragent, more preferably at least about 50 wt %, and more preferably atleast 70 wt % of the combined weight of enhancer and polar agent. Theamount is preferably at most 95 wt %, more preferably at most 80 wt %,and more preferably at most 75 wt % of the combined weight of theenhancer and polar agent. Thus, as shown in the examples, a typicaldosage form may contain a wide range of concentrations of enhancercompounds depending on the compound itself and its efficacy in enhancingthe permeability of polar agents following oral administration.Concentrations as low as 0.001% by weight up to 20% have beendemonstrated to be effective in enhancement of the permeability of polaragents.

Suitable excipients are well known to those skilled in the formulationart, and any excipient or combination of excipients known in thepharmaceutical art may be used. Examples may include flow aids,stabilizers, surface active agents, binders, dispersing agents,flavorings, taste masking agents, coatings, release control agents,water, and/or other excipients typically employed for formulation oforal dosage forms. In some embodiments, the excipient may comprise oneor more materials selected from the group consisting of microcrystallinecellulose, dicalcium phosphate, lactose, pre-gelatinized starch,carnauba wax, candelilla wax, silica, and magnesium stearate.

The compositions of this invention may in some aspects be prepared bycombining one or more polar agents with suitable amounts of either asingle permeability enhancer compound or combinations thereof andoptionally with other formulation additives/excipients, mixingthoroughly, and either tableting or filling a suitable hard shellcapsule or soft gel capsule with the resulting composition. It has beenfound that in some cases, sonicating the mixture (i.e., exposure of theneuraminidase inhibitor/enhancer mixture to ultrasonic radiation) mayincrease the efficacy of the enhancer. Common methods for sonication areknown in the art, such s as use of a probe or bath sonicator.

It has also been found that in some cases, high-energy blending of themixture (e.g., exposing the mixture to significant sheer forces) mayincrease the efficacy of the enhancer. Common methods for high-energyblending include any known in the art, such as stirrers, rotor-statordevices or colloid mills.

It has also been found that in some cases, homogenization ormicronization of the mixture (e.g., exposing the mixture to extremepressure and stress forces, including but not limited to sheer,turbulence, acceleration and impact forces) may increase the efficacy ofthe enhancer by forming an emulsion of the agent/enhancer mixture inwater. Common methods for micronization include any known in the art,such as use of a high pressure homogenizer. Such micronizationtechniques may significantly reduce the particle size of the mixture inthe formulation, providing particle sizes typically <10 um in size. Forexample, a CAPMUL® MCM L8/neuraminidase inhibitor mixture may beemulsified in about an equal weight of water. This may be done byrepeatedly squirting the mixture through a narrow orifice until anemulsion is formed, or by other emulsion-forming techniques known tothose of skill in the art. Although a roughly equal weight of watertypically works well, other proportions may also be used according tothe invention.

All such methods of sonication, high-energy blending, homogenization andmicronization may alter the viscosity of the mixture. It has been foundthat in some cases, the viscosity of the mixture is significantlyincreased, sometimes by as much as 50% or more. In some cases, anincrease in viscosity may be desirable for improved manufacturability(i.e., improved efficiency of filling solid dosage form vessels such ascapsules or soft-gels) or improved content uniformity and decreasedvariability of the mixture. In some aspects, a significant increase inviscosity may increase the efficacy of the enhancer.

In some aspects, a significant increase in viscosity may indicate asuccessful endpoint of high-energy mixing, sonication or homogenization.It has also been found that in some cases of homogenization,micronization, sonication or high-energy blending of the mixture, anendothermic reaction may accompany the increase in viscosity. In someembodiments, an endothermic reaction may indicate a successful endpointof high-energy mixing, sonication or homogenization. One exemplaryembodiment that shows this effect is a combination of CAPMUL® MCM L8with peramivir, which upon sonication under some conditions undergoes anendothermic transition during which the mixture suddenly becomessignificantly more opaque and the temperature of the mixture rapidlydrops. In some cases, for example, the transition results in atemperature drop of about 10° C. from about 65-75° C.

The resulting compositions are typically viscous liquids or paste-likesolids. Additional permeability enhancers or formulation additives caneither be added prior to sonication or after sonication of the initiallipid/agent composition.

In some embodiments, a tablet, multiparticulate dosage form, capsule, orgranule containing the composition may be coated with an enteric orpH-sensitive layer to facilitate drug composition release in thegastro-intestinal tract distal to the stomach. In some embodiments, theenteric coating or pH-sensitive layer may comprise, but is not limitedto, one or more materials selected from the group enteric polymersconsisting of cellulose acetate phthalate, cellulose acetatetrimellitate, hydroxypropyl methylcellulose acetate succinate,hydroxypropyl methylcellulose phthalate, and polyvinyl acetatephthalate; and anionic polymers based on methacrylic acid andmethacrylic acid esters.

This disclosure contemplates a formulation composition comprising atleast one polar agent, a permeability enhancer, and optionally otherexcipients in a tablet or capsule configuration with optional entericcoating. In some embodiments, such compositions are non-aqueous in thatwater is excluded as a potential excipient and the only water that ispresent is that which may be present natively or naturally in theindividual formulation components. It is also contemplated that theviscosity of liquid formulations for capsule delivery applicationsaccording to the invention will be higher than the viscosity of a 5%aqueous solution of that formulation.

The following examples are provided to describe the invention in greaterdetail. The examples are intended illustrate, not to limit, theinvention.

EXAMPLE 1 General Experimental Procedures

Permeability enhancers such as CAPMUL® MCM L8, GATTEFOSSÉ 61A throughGATTEFOSSÉ 61H compositions, glycerol,3-(N,N-dimethylpalmitylammonio)propane sulfonate (PPS), Leucine,Alanine, Gelucire 44/14, Tween 20, N-methylpiperazine, andd-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) were eachmixed with peramivir and vortexed and sonicated. For example, in thecase of CAPMUL® MCM L8, the enhancer was mixed with peramivir in amountssuch that the weight ratio of enhancer to peramivir was in the range ofabout 333:1 to about 1333:1, and such that when the mixture wassubsequently diluted in HBSS to a level at which peramivir was presentat a concentration of 15 μg/mL (0.0015%) the enhancer concentration ofthe sample was in the range of 0.5% to 2.00%, as shown in the tablebelow. Mixing was conducted by sonication (using either a bath or probesonicator) which converted the relatively low viscosity liquid mixtureto a highly viscous or paste-like composition that is a stable andnon-separating.

In like manner, other enhancer compounds were mixed with peramivir inamounts such that the weight ratio of enhancer to peramivir was in therange of about 0.7:1 to about 7000:1, and, similarly, such that when themixture was subsequently diluted to a level at which peramivir waspresent at a concentration of 15 μg/mL (0.0015%) the enhancerconcentration of the sample was in the range of 0.001% to about 10%, asshown in the table below

To evaluate the effectiveness of permeability enhancers, data wereobtained to demonstrate the ability of one or more permeability enhancercompound(s) to increase peramivir permeability using Caco-2 cellpermeability assays. The assays were performed according to the methodsdescribed by Artursson P, Palm K, Luthman K., Caco-2 Monolayers inExperimental and Theoretical Predictions of Drug Transport, Adv DrugDeliv Rev. 2001 Mar. 1; 46(1-3):27-43, and by Shah P, Jogani V, BagchiT, Misra A., Role of Caco-2 Cell Monolayers in Prediction of IntestinalDrug Absorption, Biotechnol Prog. 2006 January-February; 22(1):186-98.Assays were conducted by seeding approximately 68,000 viable Caco-2cells in 1.12 cm² Costar Transwell inserts (12-well format, 0.4 micronpore size PET membranes) in Dulbecco's Modified Eagles Medium (highglucose) supplemented with 20% fetal bovine serum, glutamine, pyruvate,non-essential amino acids, epidermal growth factor, ITS (insulin,transferrin, selenium), and penicillin/streptomycin. The cells wereincubated for 21-25 days with medium changes every 2-3 days.Transepithelial electrical resistance (TEER) readings were conducted totest the quality of the cell monolayer on the Transwell membrane. Themembranes were washed in Hank's Balanced Salt Solution (HBSS, availablefrom Mediatech, Inc., Herndon, Va.) and the resistance across themembrane was measured. Wells having TEER readings of 200 Ωcm² or higherwere used in the permeability assays.

Assays were conducted by washing the transwell inserts containing aCaco-2 cell monolayer in HBSS and placing them in 12-well plates with1.5 ml of HBSS in the lower well. The peramivir containing testformulation was diluted into HBSS to provide a peramivir concentrationof 15 μg/mL, and 0.5 ml of the solution was added to the transwellinsert. Each formulation was tested in triplicate. The transwell insertswere incubated in a 37° C. incubator with rotation at 50 rpm for 30minutes. At the end of this period the transwell inserts were placed ina fresh 1.5 ml of HBSS in a new well of the 12-well plate and incubatedfor an additional 30 minutes. A total of 8 to 10 thirty-minute timepoints were collected by sequentially moving the transwell inserts tofresh 1.5 ml HBSS in successive wells of the 12-well plates. The amountof peramivir transported into the lower wells was quantitated by LC-MSto define the rate of peramivir transported across the membrane for eachtest formulation. The reference control was composed of peramivir inHBSS in the absence of any permeability enhancer compounds.

As used herein, the term “fold increase” designates the multiplicativeeffect on peramivir permeability provided by the enhancer. Thus, thedegree of permeability enhancement may be expressed either as apercentage of the permeability of peramivir alone (in the absence of anpermeability enhancing compound or in the presence of a compound whichis ineffective in enhancing its permeability), in which case a result of100% or less indicates no enhancement in permeability. Likewise, thesevalues can be (and are in the figures and in the data sets below)reported as a “fold” value in which 1-fold is equivalent to peramiviralone (i.e., the same as 100%), a 1.5-fold value is the is same as 150%of the value for peramivir alone, a fold value of 5 is the equivalent of500% enhancement, and so forth.

EXAMPLE 2 Evaluation of Various Permeability Enhancers on Caco-2Permeability of Peramivir

As shown in FIG. 1, Caco-2 cell permeability of peramivir was enhancedwhen combined with a variety of permeability enhancers at a singleconcentration.

A significant permeability increase was provided by Capmul® MCM L8, withsignificant increases also observed using many of the Gattefossecompositions. Compared to other materials so far tested, Capmul® MCM L8seems to have additional advantages. As shown in FIGS. 2 and 3, thepermeability enhancement effect by Capmul® MCM L8 (FIG. 2) seems to besaturable and occurs at rather low levels of the enhancer by weightcompared to Gattefosse compositions (FIG. 3).

CAPMUL® MCM L8. Caco-2 permeability of peramivir combined with theCAPMUL® MCM L8 was performed as described above. The results arereported in FIG. 2 and Table 1, the former showing a visualrepresentation of the comparative fold increase and related data fromone dataset, the latter showing another dataset illustrating both theobserved rate of peramivir transport across the membrane (ng peramivirtransported/minute) and the fold increase in permeability across theCaco-2 cell membranes due to permeability enhancers, as compared to acontrol containing peramivir alone, both at a peramivir concentration of15 μg/mL.

Variation of CAPMUL® MCM L8 concentrations resulted in a significantincrease in peramivir permeability across Caco-2 cell membranes asCAPMUL® MCM L8 was increased from 0.5% to 1% in HBSS. In tests performedto date the best permeability enhancement was observed at about 1%CAPMUL® MCM L8 concentration, with slightly suppressed permeability athigher (for example, 5%) concentration. As can be seen, a significantimprovement in permeability was obtained by including CAPMUL® MCM L8 asa peramivir permeability enhancer.

GATTEFOSSÉ 61A through GATTEFOSSÉ 61H. The results obtained by includingGATTEFOSSÉ compositions 61A through 61H at concentrations varying from0.5% to 2% in an HBSS solution containing 15 μg/mL of peramivir areshown in the Table below. As can be seen, significant increases inperamivir permeability were effected by inclusion of the GATTEFOSSÉ 61Athrough 61H compounds, generally at or above an enhancer compoundconcentration of at least 0.5%. Similar results are graphically shownfor another Gattefosse dataset illustrating compositions 61A, 61E and61F in FIG. 3.

Dimethylpalmityl-Ammonio Propanesulfonate. Not all potentialpermeability enhancers have been shown to be effective for peramivir inCaco-2 cell assays. For example, Tween-80, sodium salicylate, andmethylpiperazine have been shown for the most part to be negative.Additional compounds and compositions have been tested to develop anincreased understanding of the mechanism or mechanisms that are relevantfor peramivir permeability enhancement. Promising new data also involvesphosphatidylcholine (PC) and3-(N,N-dimethylpalmitylammonio)propanesulfonate (PPS). PC has shown somepotential for peramivir permeability enhancement in Caco-2 cell assaysbut its aqueous insolubility confounds the results. PC also appears toact through a tight junction regulatory mechanism.

In contrast to the other permeability enhancing compositions that alterof tight-junction behavior, PPS acts through a different mechanism. PPSis a zwitterionic detergent and has membrane fluidizing properties thatenhance transport using transcellular permeability pathway. As shown inFIG. 4, Caco-2 cell assays with PPS varying from 0.001% to 0.01% finalconcentration provided substantially increased peramivir permeability onpar to that seen with Capmul MCM L8. This result is especiallyinteresting in comparison to the negative results with Tween-80, anon-ionic surfactant that was also anticipated to also modify membranefluidity properties. Additional tests on other non-ionic and ionicsurfactants, including bile salts, will be conducted. Although PPS isnot listed on the FDA's list of approved inactive ingredients, it is notcytotoxic in the concentrations used. Table 1 also shows thedose-response characteristics of other permeability enhancers accordingto the invention, on peramivir transport across a Caco-2 cell monolayer.As can be seen, significant permeability enhancement was obtained when3-(N,N-dimethylpalmitylammonio)propane sulfonate (PPS),

Glycerol. The ability of glycerol to enhance peramivir permeability isshown in the Table 1 below. Less than 150% peramivir permeabilityenhancement was obtained at lower glycerol concentrations at or belowabout 1%. However, a substantial increase in permeability enhancementwas obtained between when the glycerol concentration was above about 1%,suitably in the range from about 2% to as high as 10%.

The results shown in FIG. 5 demonstrate that similar to Capmul MCM L8,glycerol is ineffective in enhancing permeability at low concentrationsbut provides substantially increased permeability at higherconcentrations of up to to 5%. This result is consistent with publishedobservations that glycerol disrupts tight junction organization leadingto increased paracellular transport (Wiebe J P, Kowalik A, Gallardi R L,Egeler O, and Clubb B H (2000) “Glycerol disrupts tightjunction-associated actin microfilaments, occluding, and microtubules inSertoli cells”, J. Androl, 21, 625-635). Both the safety profile ofglycerol and its in vivo permeability enhancement capability withperamivir make it an attractive alternative for consideration in humantrials

Other permeability enhancers. Gelucire 44/14, phosphatidylcholine, Tween20, and N-methylpiperazine were used as permeability enhancers. Withsome of these substantial enhancement was obtained at very low enhancerconcentration, in some instances as low as 0.0016% (Table 1),

TABLE 1 Effect of varying permeability enhancer concentrations ontransport of peramivir across a Caco-2 cell membrane. Negative Controlng ng peramivir peramivir Fold- Enhancer transported/ transported/Enhancement Enhancer Concentration min min Over Control Capmul MCM 0.50%9.97 0.33 30.2 L8 1.00% 11.07 0.33 33.6 2.00% 10.09 0.33 30.6 Glycerol0.50% 0.16 0.14 1.14   1% 0.18 0.14 1.29   2% 0.97 0.14 6.93   5% 4.350.14 31.1   10% 5.34 0.14 38.1 Gattefosse 61A 0.50% 1.34 0.33 4.06 0  1% 1.97 0.33 5.97 1   2% 4.41 0.33 13.4 2 Gattefosse 61B 0.50% 0.960.33 2.91 3   1% 1.74 0.33 5.27 4   2% 2.16 0.33 6.55 5 Gattefosse 61C0.50% 0.33 0.33 1 6   1% 0.49 0.33 1.48 7   2% 0.87 0.33 2.64 8Gattefosse 61D 0.50% 0.68 0.33 2.06 9   1% 0.47 0.33 1.42 0   2% 0.60.33 1.82 1 Gattefosse 61E 0.50% 1.34 0.33 4.06 2   1% 2.02 0.33 6.12 3  2% 4.56 0.33 13.8 4 Gattefosse 61F 0.50% 1.31 0.33 3.97 5   1% 1.780.33 5.39 6   2% 3.3 0.33 10 7 Gattefosse 61G 0.50% 1.06 0.33 3.21 8  1% 1.52 0.33 4.61 9   2% 1.12 0.33 3.39 0 Gattefosse 61H 0.50% 1.060.33 3.21 1   1% 2.42 0.33 7.33 2   2% 5.81 0.33 17.6 3 PPS 0.001%  0.480.18 2.67 4 0.005%  1.97 0.18 10.9 5 0.010%  3.73 0.18 20.7 6 TPGS   1%9.52 0.33 28.9 7 Capmul MCM   1% 14.35 0.33 43.5 L8 + TPGS 8 Leucine0.0024%  2.2 0.57 3.86 9 Alanine 0.0016%  2.5 0.57 4.39 0 Gelucire 44/140.50% 0.75 0.33 2.27 1   1% 2.06 0.33 6.24 2   2% 2.6 0.33 7.9 3Phosphatidyl- 0.05% 0.4 0.19 2.11 4 choline 0.10% 0.4 0.19 2.11 5 0.20%1.33 0.19 7 6 Tween 20 0.50% 1.96 0.33 5.94 7   1% 3.12 0.33 9.45 8   2%3.18 0.33 9.64 9 N-Methyl- 0.50% 0.39 0.33 1.18 0 piperazine   1% 0.850.33 2.58 1   2% 0.75 0.33 2.27

EXAMPLE 3 Proposed Initial Human Pharmacokinetic Trial

This is a prophetic example. To be effective, a proposed enteric-coatedperamivir oral dosage form should contain an adequate amount of apermeability enhancer to impact either the paracellular or transcellulartransport pathways, or both. Once such a condition has been identified,the amount of peramivir can be appropriately scaled to achieve thedesired blood level. For example, the amount of permeability enhancershould take into account the volume of a human duodenum: 750-1000 mg and1500-2000 mg of enhancer should roughly correspond to the dose at thelower and upper ranges, respectively, of the proportionate volume of thehuman duodenum.

An initial human PK trial should be designed to test the utility of bothCapmul® MCM L8 and glycerol. A four- or five-way crossover protocolutilizing enteric-coated softgels is envisioned. This involves dosingsubjects with either one or two softgels in separate arms and examiningthe PK data to determine if the peramivir blood levels are doseproportional. Peramivir dose proportionality would indicate a nearsaturating effect from the lower dose of the permeability enhancer used.Alternatively, separate dosage forms can be manufactured for each armwherein peramivir is kept constant and two amounts of permeabilityenhancer is used. The following arms are proposed to both testpermeability enhancer function and to limit the number of dosage formsthat must be manufactured.

Arm 1: 150 mg peramivir, 765 mg Capmul MCM L8 in a single dosage form(765 mg of Capmul MCM L8 is the highest currently approved amount on theFDA inactive ingredient list.)

Arm 2: 300 mg peramivir, 1530 mg Capmul MCM L8 dosed as two gelcaps usedin Arm 1.

Arm 3: 150 mg peramivir, 1000 mg glycerol in a single dosage form(although 223.8 mg of glycerol is the highest currently approved amount,its safety and use as a s food additive should not present a significantregulatory barrier for increasing that limit.)

Arm 4: 300 mg peramivir, 2000 mg glycerol dosed as two gelcaps used inArm 3.

Arm 5: 150 mg or 300 mg peramivir plus inert filler in a single dosageform (this is an optional negative control arm included to scale theimpact of the permeability enhancers. It may be unnecessary depending onprior clinical experience with oral peramivir.)

It is anticipated that results from this trial will provide importantinformation to demonstrate the potential to deliver peramivir orally andto use as a guide in defining an optimized formulation and peramivirdrug load.

Although the invention is illustrated and described herein withreference to specific embodiments, the invention is not intended to belimited to the details shown. Rather, various modifications may be madein the details within the scope and range of equivalents of the claimswithout departing from the invention.

What is claimed is:
 1. An oral dosage form comprising a compositionhaving an enteric- or pH-sensitive coating or layer surrounding same,said composition comprising: a polar agent, and a permeability enhancercomprising glycerol, wherein the amount of the polar agent transportedacross a Caco-2 cell membrane by said oral dosage form is increased byat least 150% relative to the amount transported across the Caco-2 Cellmembrane in the absence of the permeability enhancer.
 2. The oral dosageform of claim 1, wherein the permeability enhancer further comprises oneor more agent selected from the group consisting of fatty acids, fattyacid esters, fatty acid salts, surfactants, cyclodextrins, sodiumsalicylate, ethylenediamine tetraacetic acid, citric acid, chitosan,chitosan derivatives, palmitoyl carnitine chloride, acyl carnitines,ethylene glycol tetraacetic acid,3-alkylamido-2-alkoxypropyl-phosphocholine derivatives,alkanoylcholines, N-acetylated amino acids, mucoadhesive polymers,phospholipids, piperine, 1-methylpiperazine, α-amino acids, and mineraloil.
 3. The oral dosage form of claim 2, wherein the fatty acid estersare fatty acid esters of glycerol, and the chitosan derivatives areN-trimethyl chitosan chloride and monocarboxymethyl-chitosan.
 4. Theoral dosage form of claim 1, wherein the permeability enhancer furthercomprises one or more agent selected from the group consisting of one ormore fatty acid esters, and dimethylpalmityl-ammonio propanesulfonate.5. The oral dosage form of claim 4, wherein the fatty acid esters aremonoglycerides of capric and/or caprylic acids, and diglycerides ofcapric and/or caprylic acids.
 6. The oral dosage form of claim 1,wherein the polar agent and the permeability enhancer form a mixturethat is emulsified.
 7. The oral dosage form of claim 1, wherein thecomposition comprises a therapeutically effective amount of the polaragent and a permeability-enhancing amount of said permeability enhancer.8. The oral dosage form of claim 7, wherein the permeability enhancerfurther comprises one or more agent selected from the group consistingof fatty acids, fatty acid esters, fatty acid salts, surfactants,cyclodextrins, sodium salicylate, ethylenediamine tetraacetic acid,citric acid, chitosan, chitosan derivatives, palmitoyl carnitinechloride, acyl carnitines, ethylene glycol tetraacetic acid,3-alkylamido-2-alkoxypropyl-phosphocholine derivatives,alkanoylcholines, N-acetylated amino acids, mucoadhesive polymers,phospholipids, piperine, 1-methylpiperazine, α-amino acids, and mineraloil.
 9. The oral dosage form of claim 8, wherein the fatty acid estersare fatty acid esters of glycerol, and the chitosan derivatives areN-trimethyl chitosan chloride and monocarboxymethyl-chitosan.
 10. Theoral dosage form of claim 7, wherein the permeability enhancer furthercomprises one or more agent selected from the group consisting of one ormore fatty acid esters, and dimethylpalmityl-ammonio propanesulfonate.11. The oral dosage form of claim 10, wherein the fatty acid esters aremonoglycerides of capric and/or caprylic acids, and diglycerides ofcapric and/or caprylic acids.
 12. The oral dosage form of claim 7,wherein the polar agent and the permeability enhancer form a mixturethat is emulsified.
 13. The oral dosage form of claim 7, wherein thepermeability enhancer is present in the composition at a concentrationfrom 5% to 95% of the combined weight of the polar agent and thepermeability enhancer.
 14. The oral dosage form of claim 7, wherein theoral dosage form is an encapsulated gel comprising an outer gel capcontaining the composition.
 15. The oral dosage form of claim 7, whereinthe polar agent and permeability enhancer are mixed by sonication.
 16. Amethod for treating or preventing influenza infection in a subject inneed thereof, said method comprising administering an effective amountof a composition according to claim 1 to said subject.
 17. The method ofclaim 16, wherein the permeability enhancer further comprises one ormore agent selected from the group consisting of fatty acids, fatty acidesters, fatty acid salts, surfactants, cyclodextrins, sodium salicylate,ethylenediamine tetraacetic acid, citric acid, chitosan, chitosanderivatives, palmitoyl carnitine chloride, acyl carnitines, ethyleneglycol tetraacetic acid, 3-alkylamido-2-alkoxypropyl-phosphocholinederivatives, alkanoylcholines, N-acetylated amino acids, mucoadhesivepolymers, phospholipids, piperine, 1-methylpiperazine, α-amino acids,and mineral oil.
 18. The method of claim 17, the fatty acid esters arefatty acid esters of glycerol, and the chitosan derivatives areN-trimethyl chitosan chloride and monocarboxymethyl-chitosan.
 19. Themethod of claim 16, wherein the permeability enhancer further comprisesone or more agent selected from the group consisting of one or morefatty acid esters, and dimethylpalmityl-ammonio propanesulfonate. 20.The method of claim 19, wherein the fatty acid esters are monoglyceridesof capric and/or caprylic acids, and diglycerides of capric and/orcaprylic acids.